Ribosome biogenesis is an essential but complex multistep pathway which exists in all living cells. The precursor rRNA (pre-rRNA) encodes three distinct RNAs, but is transcribed as a single precursor molecule that must be correctly modified, folded, processed and assembled with proteins in order to yield the two subunits that together form a mature ribosome. The focus of my research has been to examine and identify cis-acting elements and trans-acting factors which are critical for the processing events of pre-rRNA processing, and thus essential for cell survival. Over the past year my lab has made progress on two fronts. First, we are using the genetics available in the yeast, S.cerevisiae to examine a proposed intramolecular interaction necessary for pre-rRNA processing. Second, we are using biochemical methods to identify proteins which comprise the U8 small nucleolar ribonucleoprotein particle (U8 snoRNP), an essential trans-acting factor required for accumulation of newly formed large ribosomal subunits.Previously I described a model describing the mechanisms by which U8 snoRNA appeared to facilitate pre-rRNA processing in the Xenopus oocyte (1). This model predicted a specific intramolecular interaction in pre-rRNA. Formation of this stem should be critical for pre-rRNA processing. Because of the complexity of the Xenopus oocyte and the many different aspects of RNA processing addressed by this model, I used the yeast system to directly test a smaller aspect of this model. The genetic and biochemical manipulations which are possible in yeast made it possible to directly test and analyze specific aspects of the model proposed in Xenopus. The experiments in yeast unequivocally demonstrated that formation of this intramolecular interaction is critical for pre-rRNA processing (2). Over the past year additional experiments in yeast have implicated other cis-acting elements, which may act either as primary sequences or secondary structures, that also play an important roles in processing. We have begun to undertake more detailed mutagenesis of this region of the rRNA to identify those elements that affect the efficiency or the accuracy of pre-rRNA processing in yeast. The data generated in the yeast studies will later be applied to parallel experiments in Xenopus, which to date is the only existing model system for examining rRNA processing in vertebrates.We are continuing our characterization of trans-acting factors essential for pre-rRNA processing in vertebrates. I previously demonstrated that U8 snoRNP is essential for pre-rRNA processing in Xenopus oocytes. In the absence of U8 RNA, pre-rRNA processing is inhibited and no mature rRNA will accumulate (1). Mutageneis of U8 RNA indicated that U8 RNA was necessary, but not sufficient to direct pre-rRNA processing; proteins were present that affected the stability of the U8 RNP and the efficiency of processing (1). To better understand how the U8 proteins contribute to stability of U8 RNA and how they affect in vivo function of U8 RNP, we have been identifying proteins which specifically bind U8 RNA in vitro. In the past year we have identified and partially characterized a 29 kDa protein in Xenopus ovary extracts which binds U8 RNA. This protein binds U8 RNA with high specificity and can be crosslinked to U8 (3). In vitro competition binding assays indicated this protein is unique to U8 and does not represent a common shared protein present on all snoRNPs. Preliminary protein analysis indicates this novel protein represents the first identified constituent of the U8 RNP, the only vertebrate snoRNP required for processing of 5.8S and 28S rRNA. Future work will continue to characterize the X29 protein and identify other putative U8 RNA binding proteins. By taking advantage of the two different model systems, yeast and Xenopus, we hope to better understand the basic mechanisms of pre-rRNA processing and to identify and the cis- and trans-acting components involved. Identification of common components as well as species specific elements will help us understand the basic mechanisms at play in the universal process of pre-rRNA maturation.1) Peculis, B. A. (1997) The sequence of the 5 end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation. Mol Cell Biol 17:3702-3713.2) Peculis, B.A. and Greer, C.L. (1998) The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast. RNA 4:1610-1622.3) Tomasevic, N. and Peculis, B.A. Identification of a U8-specific binding protein. Submitted.